This program project will continue to focus on antigen presentation and T cell recognition in patients with multiple sclerosis. During the past three years considerable efforts were directed at establishing and validating new technology, now in place for the continuation of the PPG. These novel approaches will be used to visualize antigen presentation in the central nervous system and to molecularly define the T cell and B cell populations involved in the disease process. Project 1 has developed a novel approach for the generation of MAC class II terriers, which will be used to quantify and characterize antigen-specific COD T cells in MS patients and a humanized mouse model of the disease. Project 2 has isolated expanded populations of T cells from the cerebrospinal fluid of patients with MS and will characterize the antigen receptors of T cell and B cell populations. Combinatorial peptide libraries and MHC class II terriers will be used to define the antigen specificity of T cell clones established from the cerebrospinal fluid. B cells will be isolated from cerebrospinal fluid by FACES and MS plaque tissue by laser capture micro dissection, so that recombinant antibodies can be expressed based on the isolated heavy and light chain sequences. Such recombinant antibodies provide an abundant source of these proteins for definition of their target specificity, to be established by a polemics approach, and assessment of pathogenicity. Project 3 will examine the entry of antigen presenting cells into the CNS using a newly created mouse model in which a GFP moiety is attached to the MHC class II beta chain through knock-in technology. The interaction of these GFP expressing antigen presenting cells with infiltrating T cells will be investigated in the optic nerve of live animals by 2-photon microscopy. Each of the investigators contributes a unique area of expertise and innovative approaches to this important clinical problem. The resulting program is highly synergistic and will allow the application of powerful new tools in proteomics and biochemistry to the pathophysiology of human inflammatory disease.